R E S E A R C H
Open Access
Antimycobacterial and cytotoxicity activity
of microcystins
Daniela Fernandes Ramos
1*, Alexandre Matthiensen
2, Wilson Colvara
3, Ana Paula Souza de Votto
4,
Gilma Santos Trindade
4, Pedro Eduardo Almeida da Silva
1and João Sarkis Yunes
5Abstract
Background: The present work aimed to evaluate the antimycobacterial activity and cytotoxicity of Microcystis aeruginosa toxins, the MC-LR variant and purified extract of [D-Leu1
] microcystin-LR.
Methods: The antimicrobial activity of M. aeruginosa extract and microcystin was evaluated by resazurin microtiter assay against Mycobacterium tuberculosis, M. terrae, M. chelonae and M. kansasii. The cytotoxicity assay was
performed by trypan blue exclusion against the HTC cell line.
Results: Antimicrobial activity was observed in the hexanic extract of M. aeruginosa (RST 9501 strain) against M. tuberculosis, including sensitive and resistant strains with minimal inhibitory concentrations (MIC) between 1.93 μM and 0.06μM. The high activity of M. aeruginosa hexanic extract could be attributed to the major presence of the toxins MC-LR and [D-Leu1] MC-LR that showed activity at MIC between 53 and 0.42μM against tested mycobacterial strains. Even at the highest concentration tested, no toxicity of M. aeruginosa extracts was identified against HTC cells.
Conclusions: These preliminary results suggest that [D-Leu1] MC-LR is a promising candidate for the development of a new antimycobacterial agent.
Keywords: Mycobacteria, Antimycobacterial agents, Cytotoxic activity, Microcystins Background
“Nontuberculous mycobacteria” is a general expression ap-plied for different species of the genus Mycobacterium that do not belong to the Mycobacterium tuberculosis complex [1]. They are also recognized as causative agents of oppor-tunistic infections in humans that affect mainly patients with preexisting pulmonary diseases– such as chronic ob-structive pulmonary disease or tuberculosis (TB)– or those with impaired systemic immunity [2-4]. The latter group includes patients with HIV infection, leukemia and under immunosuppressive therapy [5,6].
For most nontuberculous mycobacterial infections, treatment is based on drugs that may differ according to the causal agent, in particular between slow- (e.g. M. avium, M. kansasii) and fast-growing species (e.g. M. abscessus, M. fortuitum) [1]. In general, drug therapy is long, costly, and often associated with toxic side effects.
In addition, high rates of natural antibiotic resistance are common among nontuberculous mycobacteria, which increases the challenges for new drug discovery [1,7].
TB remains a major global health problem reaching millions of people every year and ranking as the second leading cause of death among infectious diseases world-wide [8]. The current treatment available against TB es-tablishes a multidrug regimen that lasts a minimum of six months and does not guarantee a complete eradica-tion of the infeceradica-tion [9].
Furthermore, the increased number of TB cases due to multidrug resistant and extensively drug resistant strains (MDR and XDR) and HIV co-infection have pointed out the urgent need for alternative treatment. In recent years, research on the development of new anti-TB ther-apies has focused on novel agents from both synthetic and natural sources [10]. For thousands of years, plant-derived drugs have been empirically used in the treat-ment of numerous human disorders. Many conventional drugs originate from plant sources, such as aspirin (from willow bark), digoxin (from foxglove), quinine (from * Correspondence:daniferamos@gmail.com
1
Research Center in Medical Microbiology, Federal University of Rio Grande (FURG), Rio Grande, Rio Grande do Sul State, Brazil
Full list of author information is available at the end of the article
© 2015 Ramos et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
cinchona bark), and morphine (from the opium poppy) [11].
Marine natural products play an important role in drug development particularly in anticancer, antibiotic and antiparasitic therapies. It is well known that macrocyclic peptides may demonstrate drug-like physi-cochemical and pharmacokinetic properties such as good metabolic stability, solubility, lipophilicity and bioavailability [12]. More than 800 secondary metabo-lites belonging to several classes of substances have been isolated and identified, which includes enzyme in-hibitors; photosynthesis inin-hibitors; antimicrobial, anti-mitotic, immunosuppressive and antitumor peptides [13-15].
Microcystins, more than 65 structural variants are cur-rently known, are cyclic heptapeptides, composed of seven amino acids, namely, five non-protein and two protein amino acids. These two protein amino acids distinguish microcystins from one another, while the other amino acids are more or less constant among the variants [16-18].
Structural variations have been identified at all seven positions of the heptapeptide ring. Microcystin-LR (MC-LR) (Figure 1) is the most commonly identified cyanotoxin in environmental samples, which presents a leucine (L) and an arginine (R) respectively in X and Y positions of the cyclic heptapeptide [19,20]. The com-bined presence of the two L-amino acids is used in the nomenclature of the variants while the position 1, which contains D-Ala, is relatively conserved [21].
Another variant of microcystin is [D-Leu1] MC-LR,
which contains D-Leu in position 1. This toxin was de-tected in cells of Microcystis aeruginosa (RST 9501 strain) isolated from Patos Lagoon (southern Brazil). This major waterbody has a history of extensive nuis-ance blooms and scums of hepatotoxic Microcystis [22].
The present study evaluated the antimycobacterial ac-tivity and cytotoxicity of Microcystis aeruginosa toxins,
the variant MC-LR and purified extract of [D-Leu1]
microcystin-LR against Mycobacterium tuberculosis, M. chelonae, M. terrae and M. kansasii.
Methods
Microcystis culture conditions
Microcystis RST 9501 (UPC Culture Collection, Federal University of Rio Grande) isolated from the estuary of
Patos Lagoon is the [D-Leu1] MC-LR producing variant
and was grown in BG-11 medium with nitrate as previ-ously described [23,24].
Preparation of microcystis aeruginosa extracts
The extract was prepared using lyophilized cells of Microcystis aeruginosa according to the protocol de-scribed by Beattie et al. [24]. Briefly, the cells were dis-solved in absolute methanol (Sigma, USA), sonicated three times and centrifuged (10,000 × g) at 4°C, for ten minutes. Extracts were evaporated at 40°C and then redissolved in ultrapure water (Direct Q3, Millipore, USA). The other extract preparations, presented in Table 1, replaced methanol with hexane, chloroform or water. Finally, samples were centrifuged and the super-natant was collected and stored at-20°C. Microcystin content was determined using a commercial enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies (EnviroLogix Inc., USA). Different concen-trations of microcystin were prepared after appropriate
dilutions with phosphate buffered saline (PBS – Ca+2
and Mg+2free). Characterization of microcystins produced by the strain RST 9501 was previously reported by Mat-thiensen et al. [17,25]. For the extraction of microcystin
from cells of the strain RST 9501, the toxin [D-Leu1]
microcystin-LR was purified from cell extracts, following Lawton et al. [26]. The chemical compound microcystin-LR was purchased from Sigma (USA).
Finally, both toxins were resuspended in water and then analyzed by high performance liquid
chromatog-raphy (HPLC – Shimadzu SCL-10Avp, Japan) to
deter-mine the concentration of microcystins prior to tests.
Microcystin analysis
Analysis of microcystin obtained from Microcystis RST 9501 was performed using the HPLC equipment Shimadzu
Figure 1 General structure of microcystins (LR) with leucine (L) in the amino acid position 2 and arginine (R) in the amino acid position 4 and the structural differences in the position 1 of MC-LR (left) and [D-Leu1] MC-LR (right).
SCL-10AVP(Japan). The analysis was carried out using a
C18Luna (4.6 × 250 mm, 5μm particle size; Phenomenex,
USA) reversed-phase column at 40°C with UV detection at 238 nm. The mobile phase was Milli-Q water/CH3CN (J. T. Baker, USA), both containing 0.05 % (v/v) trifluoroacetic acid (Merck, Germany), initially at 65:35 and using a linear gradient over 20 minutes of 100 % CH3CN at 1 mL.min-1.
Isolates and mycobacterium spp. preparation
The antimicrobial activity of Microcystis aeruginosa extract and microcystin were evaluated against Mycobacterium tu-berculosis H37Rv (ATCC 27294) pan-susceptible strain and against two clinical isolate mono-resistant to isoniazid and rifampicin with katG S315T and rpoB S531L respectively. Furthermore, M. aeruginosa extract, [D-Leu1] MC-LR, and microcystin-LR (Sigma, USA) toxins were tested against the nontuberculous mycobacteria: M. terrae (ATCC15755), M. chelonae (ATCC 946) and M. kansasii (ATCC12478).
The bacterial suspensions obtained of culture in Ogawa Kudoh medium for about 14 days were prepared in sterile water containing pearls of glass of 3 mm. The suspension was homogenized by vortex agitation and the turbidity was adjusted in agreement with tube one of the scale of McFar-land (3.2 × 107cfu/mL). The inoculum was prepared dilut-ing the bacterial suspension in the proportion of 1:25 in medium 7H9 broth [4.7 g of Middlebrook 7H9 broth base (BD Difco, USA) 2 mL of glycerol (Vetec, Brazil) in 900 mL of water] enriched with 10 % oleic acid-albumin-dextrose-catalase (OADC– BBL, Media Additives, USA) [27].
Evaluation of antimycobacterial activity
The method used for the determination of the antimyco-bacterial activity was the resazurin microtiter assay [28]. In brief, the assay is accomplished on microplates (96 wells) using resazurin as indicator of cellular viability. Medium 7H9 enriched with 10 % OADC was employed. The extracts and pure microcystin were weighed, dis-solved in DMSO and the determination of minimal in-hibitory concentration (MIC) was carried out starting from 53 to 0.06μM in serial dilutions of 1:2.
Cytotoxicity assay
The HTC cell line was obtained from the Cell Bank of Rio de Janeiro, Brazil. HTC cells were grown in RPMI 1640 medium (Gibco, USA) supplemented with sodium bicar-bonate (0.2 g/L) (Vetec, Brazil), L-glutamine (0.3 g/L) (Vetec, Brazil), Hepes (25 mM) (Acros, USA) and b-mercaptoethanol (5 × 10–5 M) (Sigma, Germany), with 10 % fetal bovine serum (Gibco, Brazil), 1 % of antibiotic
and antimycotic (penicillin – 100 U/mL,
streptomycin-100 mg/mL and amphotericin B - 0.25 mg/mL), in dispos-able plastic flasks, at 37°C.
The cytotoxicity assay was performed by trypan blue exclusion after 24 hours of incubation with microcystins. Three independent experiments were carried out using triplicates in each experiment. Data are expressed as mean + standard error and analyzed using ANOVA followed by Tukey’s multiple range test. Significance level was fixed in 0.05.
Results
M. aeruginosa RST 9501 extracts were evaluated against M. tuberculosis pan-susceptible (H37Rv), rifampicin-(RIFr) and isoniazid-resistant strains (INHr). The MIC
for these four extracts ranged from 1.93μM to 0.06 μM.
The aqueous and chloroformic extracts did not present antimycobacterial activity within these concentrations.
The methanolic extract had a MIC of 1.93 μM against
H37Rv and of 0.96 μM against the tested resistant
strains. The hexanic extract showed the highest activity,
with a MIC of 0.12μM against INHr, ≤ 0.60 μM against
RIFr and≤ 0.06 μM against H37Rv (Table 1). This high
activity of the hexanic extract could be attributed to the possible greater concentration of lipophilic compounds. Therefore, the molecules of microcystins (MC-LR and [D-Leu1] MC-LR) were evaluated against M. tuberculosis H37Rv, RIFr and INHr strains.
The cyanotoxin MC-LR did not present any inhibitory activity on the three strains at the concentration of
53μM. On the other hand, [D-Leu1] MC-LR was active
with a similar MIC (13.2 μM) for susceptible and resist-ant M. tuberculosis strains (Table 2). Therefore, the iso-lated cyanotoxin showed a MIC up to 220 times higher than that of the hexanic extract.
The two microcystins were evaluated against three nontuberculous mycobacteria showing high activity for all species tested. M. terrae was the most resistant to nontuberculous mycobacteria, it showed antimycobac-terial activity against the two tested toxins with MIC of
6.74μM and 1.08 μM for [D-Leu1] MC-LR and MC-LR,
respectively (Table 3).
A further comparison between both toxins effects sug-gested that microcystin MC-LR showed lower activity than [D-Leu1] MC-LR against M. chelonae and M. kansasii, with
a minimum inhibitory concentration of 2.15 μM for the
Table 1 Minimum inhibitory concentration (MIC) of different Microcystis aeruginosa extracts against Mycobacterium tuberculosis strains
Extracts M. tuberculosis H37Rv RMPr INHr (μM) (μM) (μM) M. aeruginosa (aqueous) R >1.93 R >1.93 R >1.93 M. aeruginosa (hexanic) S ≤0.06 S ≤0.60 S 0.12 M. aeruginosa (chloroformic) R >1.93 R >1.93 R >1.93 M. aeruginosa (methanolic) S 1.93 S 0.96 S 0.96
R: resistant, S: sensitive, H37Rv: sensitive strain, RMPr: rifampicin-resistant strain,
INHr: isoniazid-resistant strain. Extract with MIC > 1.93μM were considered inactive.
two strains, while the other variant showed minimum in-hibitory concentration of 0.84μM and 0.42 μM for M. che-lonae and M. kansasii, respectively.
Exposure of HTC cells to Microcystis aeruginosa strain
9501 which produces [D-Leu1] MC-LR induces a decrease
in viable cell number, as determined by trypan blue exclu-sion, in a concentration dependent manner, 24 hours after exposure (Figure 2). There were no differences in the num-ber of viable cells (p > 0.05) between control and treated
cells in the lowest concentration (96.36 μM). However,
from the concentration of 192.71μM on, there was signifi-cant difference (p < 0.05) in the number of viable cells, which indicates cytotoxic effect. Note that at the highest concentration (578.15μM) no viable cell was found.
Discussion
In recent years, there has been a growing interest in the study of natural peptide molecules, whose mode of ac-tion promises both low susceptibility to multidrug resist-ance mechanisms and high activity against a vast range of microorganisms [29,30]. The application of peptides for drug discovery is merited because of their ease of synthesis, large structural diversity and commonly high potency [31].
The present study identified in extracts from M. aeru-ginosa RST 9501 active compounds with antimycobac-terial activity against M. tuberculosis growth, including sensitive and resistant strains. Several metabolites pro-duced by cyanobacteria have been identified in the lit-erature. There is a special interest, because these secondary metabolites are not only disease causing
agents but also bioactive molecules applied to further studies [19,32-34]. Some works have identified a range of cyanobacteria compounds isolated from toxic blooms of Microcystis, Anabaena and Nostoc known to produce a diverse array of bioactive compounds exhibiting a broad spectrum of activity, including anticancer, anti-viral, antibacterial, antifungal and anti-inflammatory ac-tivity, besides cytotoxic activities [19,32,34,35].
In this study, four M. aeruginosa extracts were tested, the methanolic extract was more active against resistant strains (RIFr and INHr) than sensitive M. tuberculosis strains. Microcystins are a group of chemically related cyclic peptides [13] and most commonly studied group of cyanotoxins,. Therefore, in order to identify the pos-sible active compounds derived from the hexane extrac-tion that was more active among the extracts from M. aeruginosa cells evaluated, the two known cyanotoxins
([D-Leu1] MC-LR and MC-LR) were evaluated against
M. tuberuculosis and three nontuberculous mycobacteria species.
Between the two variants derived from the same cyanobacterial toxin, just [D-Leu1] MC-LR showed anti-microbial activity against three different strains of M.
Table 2 Minimum inhibitory concentration (MIC) of the microcystin variant [D-Leu1] MC-LR from extracts of M. aeruginosa RST 9501 and MC-LR (commercially available) against M. tuberculosis strains
Microcystin variant M. tuberculosis H37Rv (μM) RMPr (μM) INHr (μM) [D-Leu1] MC-LR S 13.2 S 13.2 S 26.5 MC-LR R >53 R >53 R >53 [D-Leu1
] MC-LR: microcystin isolated from the extract of Microcystis RST 9501; MC-LR: commercially obtained; R: resistant; S: sensitive; H37Rv: sensitive strain;
INHr: isoniazid-resistant strain; RMPr: rifampicin-resistant strain. Extracts with MIC > 53μM were considered inactive.
Table 3 Minimum inhibitory concentration (MIC) of the microcystin variant from extract of the M. aeruginosa RS9501 and MC-LR (obtained commercially) against M. tuberculosis strains
Microcystin Variant
Nontuberculous Mycobacteria
M. terrae (μM) M.chelonae (μM) M.kansasii (μM)
[D-Leu1] MC-LR S 6.74 S 0.84 S 0.42
MC-LR S 1.08 S 2.15 S 2.15
[D-Leu1] MC-LR: Microcystin isolated of extract of the Microcystis RST 9501 strain, MC-LR: obtained commercially, R: resistant, S: sensible.
Figure 2 Number of HTC viable cell by trypan blue exclusion test 24 hours after exposure to different concentrations of Microcystis aeruginosa strain 9501 (578.15 to 93.36 μM) which produces [D-Leu1] MC-LR. Data are expressed as mean + standard error. *Indicates significant difference from the control (p < 0.05).
tuberculosis. However, the MIC of this variant was higher than that of the methanolic and hexane extracts. According to Pauli et al. [36], the differences between the extract and pure cyanotoxin activity can be attributable to the MIC of the crude extract, which may not be a reliable antimycobac-terial activity indicator, since it, the activity may be to an-tagonist between the substances, or otherwise, synergism between them, that decreasing or increasing effects on the MIC. Moreover, an extract with high activity (lower MIC) could have several compounds with moderate antimyco-bacterial activity; becoming it the most active extract with a pure and isolated substance.
The activity of [D-Leu1] MC-LR was unchanged
against strains of M. tuberculosis resistant to rifampicin and isoniazid compared to pan-susceptible strains. This is an important finding since the resistance to isoniazid and rifampicin comprise a major drawback of tubercu-losis control programs.
Interestingly, the small antimicrobial activity of the two studied microcystins variants observed against different strains of M. tuberculosis was not observed for nontubercu-lous mycobacteria. MC-LR was less active than [D-Leu1] MC-LR against M. chelonae and M. kansasii. A few studies have shown that drug susceptibility of nontuberculous mycobacteria are distinct from that observed in M. tubercu-losis because, in general, the resistance of nontuberculous mycobacteria is related to cell wall permeability and efflux pumps, specially in the presence of a specific mutation [37]. In this study, however, M. terrae was less susceptible against cyanotoxins. In addition, the MIC against M. tuber-culosis strains was higher than that used against all nontu-berculous mycobacteria.
The activity of these molecules against M. chelonae is significant, since it indicates that the low permeability limits the activity of this kind of hydrophilic molecules, which impairs treatment [38]. Regarding the mechanism of action proposed for microcystins is the inhibiting serine/threonine phosphatases 1 and 2A, which leads to protein phosphorylation and the consequence is cyto-skeletal damage, liver necrosis and hemorrhage in the liver which is directly related to their cytotoxicity and tumor promoting activity [39]. However, there is only a few data on tissue concentrations of microcystins in posed humans or animals, which were obtained after ex-posure to high toxic doses of microcystins [40].
In our study, a single difference in the chemical consti-tution of the heptapeptide was significantly important in increasing the antimycobacterial activity. In addition, considering that leucine is a hydrophobic amino acid, this characteristic may interfere in the activity of the peptide that was enhanced by presence.
According to Mandal et al. [41], variations of amino acid residues in peptides have received considerable at-tention since they alter the activity against pathogenic
microorganisms, which has a significant impact on anti-bacterial activity [41]. A study showed a difference in their activity against M. tuberculosis and Mycobacterium avium-intracellulare by the change of only one amino acid residue in their peptide moiety [42].
Peptides may adopt secondary structures, which are responsible for their receptor affinity and biological ac-tivity. The rational design can be sufficient to endow antibacterial efficacy and to circumvent drawback effects in this potential therapeutic agent [30,31].
According to Votto et al. [43] and considering that microcystins may provoke oxidative stress, the difference in sensitivity of MDR and non-MDR cells can be associated with dissimilar antioxidant defenses. In this context, the higher catalase activity observed in the same work may help to explain, at least partially, the resistance of MDR cells to microcystin exposure. This MDR cell line with higher cata-lase activity also showed lower DNA damage than its par-ental cell line, suggesting the involvement of reactive oxygen species (ROS) in the toxicity exerted by this cyano-toxin. Therefore, the significant increase in ROS production observed in non-MDR cells, in contrast with MDR cells when both cell lines were exposed to microcystins, suggest that MDR cells, at least in part, were more resistant to microcystins due to a higher antioxidant competence. These authors also present other factors which may have contributed for the resistance to microcystin in MDR cells [43].
The choice of the cell line used in this study was sup-ported by the fact that it is in the liver that microcystins are metabolized. The results show a significant sensitiv-ity of tumoral liver cells to this substance in only 24 hours of exposure.
Conclusions
In the present study, the antimicrobial activity of the hexanic extract from M. aeruginosa RST 9501 against M. tuberculosis – including sensitive and resistance
strains, and nontuberculous mycobacteria – was
ob-served and possibly associated with the presence of cya-notoxins. When the activity of these toxins was assessed, the variant [D-Leu1] MC-LR was the most active against tested mycobacterial strains. Moreover, was not identi-fied cytotoxic activity at concentrations whose anti-microbial activity. Also, was not identified cytotoxic activity at concentrations which antimicrobial activity was observed. These results showed the importance of detailed studies on the activity of extracts and toxins de-rived from M. aeruginosa strains as promising bioactive molecules in the treatment of mycobacterial diseases.
Ethics committee approval
The present study was approved by the Ethics Commit-tee of the Federal University of Rio Grande.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DFR and PEAS conceived the study and carried out the antimycobacterial activity tests. AM, WC and JSY carried out the preparation and analysis of Microcystis aeruginosa extracts. APSV and GST conducted cytotoxicity assays. All authors read and approved the final manuscript.
Acknowledgments
The authors would like to thank CNPq and FAURG for the funding of this research.
Author details
1Research Center in Medical Microbiology, Federal University of Rio Grande (FURG), Rio Grande, Rio Grande do Sul State, Brazil.2Brazilian Corporation of Agricultural Research (Embrapa), Concórdia, Santa Catarina State, Brazil. 3
School of Chemistry and Food, Federal University of Rio Grande (FURG), Rio Grande, Rio Grande do Sul State, Brazil.4Graduation Program in Physiological Sciences, Institute of Biological Sciences, Federal University of Rio Grande (FURG), Rio Grande, Rio Grande do Sul State, Brazil.5Laboratory of
Cyanobacteria and Phycotoxins, Institute of Oceanography, Federal University of Rio Grande (FURG), Rio Grande, Rio Grande do Sul State, Brazil.
Received: 25 August 2014 Accepted: 6 March 2015
References
1. Falkinham JO. The changing pattern of nontuberculous mycobacterial disease. Can J Infect Dis. 2003;14(5):281–6.
2. van Ingen J, Boeree MJ, van Soolingen D, Mouton JW. Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria. Drug Resist Updat. 2012;15(3):149–61.
3. Oliva VM, Cezário GAG, Cocato RA, Marcondes-Machado J. Pulmonary tuberculosis: hematology, serum biochemistry and the relation with the disease duration. J Venom Anim Toxins incl Trop Dis. 2008;14(1):71–81 [http://www.scielo.br/scielo. php?pid=S1678–91992008000100006&script=sci_arttext].
4. Dias-Baptista IMF, Uso SMRS, Marcondes-Machado J. Trends in multidrug-resistant tuberculosis. J Venom Anim Toxins incl Trop Dis. 2008;14(2):203–23 [http://www. scielo.br/scielo.php?script=sci_arttext&pid=S1678–
91992008000200003&lng=en&nrm=iso&tlng=en].
5. Al-Mahruqi SH, van Ingen J, Al-Busaidy S, Boeree MJ, Al-Zadjali S, Patel A, et al. Clinical relevance of nontuberculous mycobacteria, Oman. Emerg Infect Dis. 2009;15(2):292–4.
6. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007;175(4):367–416.
7. Brown-Elliott BA, Nash KA, Wallace Jr RJ. Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria. Clin Microbiol Rev. 2012;25(3):545–82.
8. World Health Organization. Global Tuberculosis Report 2012. Geneva, Switzerland: World Health Organization; 2012.
9. Mitchison DA. Prevention of drug resistance by combined drug treatment of tuberculosis. Handb Exp Pharmacol. 2012;211:87–98.
10. Raviglione MC, Smith IM. XDR tuberculosis–implications for global public health. N Engl J Med. 2007;356(7):656–9.
11. Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress. J Antibiot (Tokyo). 2009;62(1):5–16.
12. Peña S, Scarone L, Manta E, Stewart L, Yardley V, Croft S, et al. Synthesis of a Microcystis aeruginosa predicted metabolite with antimalarial activity. Bioorg Med Chem Lett. 2012;22(15):4994–7.
13. Kehr JC, Picchi DG, Dittmann E. Natural product biosyntheses in cyanobacteria: a treasure trove of unique enzymes. Beilstein J Org Chem. 2011;7:1622–35.
14. Tan LT. Bioactive natural products from marine cyanobacteria for drug discovery. Phytochemistry. 2007;68(7):954–79.
15. Volk RB, Furkert FH. Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth. Microbiol Res. 2006;161(2):180–6.
16. Luukkainen R, Namikoshi M, Sivonen K, Rinehart KL, Niemelä SI. Isolation and identification of 12 microcystins from four strains and two bloom samples of Microcystis spp.: structure of a new hepatotoxin. Toxicon. 1994;32(1):133–9.
17. Matthiensen A, Beattie KA, Yunes JS, Kaya K, Codd GA. [D-Leu1] Microcystin-LR, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil. Phytochemistry. 2000;55(5):383–7.
18. Lemes GAF, Kist LW, Bogo MR, Yunes JS. Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from the sediments of the Patos Lagoon estuary, Brazil. J Venom Anim Toxins incl Trop Dis. 2015;21:4. doi:10.1186/s40409–015–0001–3.
19. Prasanna R, Sood A, Jaiswal P, Nayak S, Gupta V, Chaudhary V, et al. Rediscovering cyanobacteria as valuable sources of bioactive compounds. Prikl Biokhim Mikrobiol. 2010;46(2):133–47.
20. Funari E, Testai E. Human health risk assessment related to cyanotoxins exposure. Crit Rev Toxicol. 2008;38(2):97–125.
21. Sivonen K, Jones G. Cyanobacterial toxins. In: Chorus L, Bartram J, editors. Toxic cyanobacteria in water: A guide to their public health consequences, monitoring and management. London: World Health Organization and E&FN Spon; 1999. p. 41–111.
22. Chong MW, Gu KD, Lam PK, Yang M, Fong WF. Study on the cytotoxicity of microcystin-LR on cultured cells. Chemosphere. 2000;41(1–2):143–7. 23. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G. Purification and properties
of unicellular blue-green algae (order Chroococcales). Bacteriol Rev. 1971;35 (2):171–205.
24. Beattie KA, Kaya K, Sano T, Codd GA. Three dehydrobutyrine-containing microcystins from Nostoc. Phytochemistry. 1998;47(7):1289–92.
25. Matthiensen A, Yunes JS, Codd GA. Ocorrência, distribuição e toxicidade de cianobactérias no estuário da Lagoa dos Patos. RS Rev Bras Biol. 1999;59 (3):361–76.
26. Lawton LA, Edwards C, Codd GA. Extraction and high-performance liquid chromatographic method for the determination of microcystins in raw and treated waters. Analyst. 1994;119(7):1525–30.
27. Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol. 1998;36(2):362–6.
28. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002;46(8):2720–2.
29. Silva NC, Sarmento B, Pintado M. The importance of antimicrobial peptides and their potential for therapeutic use in ophthalmology. Int J Antimicrob Agents. 2013;41(1):5–10.
30. Rotem S, Mor A. Antimicrobial peptide mimics for improved therapeutic properties. Biochim Biophys Acta. 2009;1788(8):1582–92.
31. Jamieson AG, Boutard N, Sabatino D, Lubell WD. Peptide scanning for studying structure-activity relationships in drug discovery. Chem Biol Drug Des. 2013;81(1):148–65.
32. Madhumathi V, Deepa P, Jeyachandran S, Manoharan C, Vijayakumar S. Antimicrobial activity of cyanobacteria isolated from freshwater lake. Int J Microbiol Res. 2011;2(3):213–6.
33. Jaiswal P, Prasanna R, Singh PK. Characterization of the biocidal spectrum of extracellular filtrates of Microcystis aeruginosa. Indian J Microbiol. 2011;51 (4):509–14.
34. Jaiswal P, Singh PK, Prasanna R. Cyanobacterial bioactive molecules– an overview of their toxic properties. Can J Microbiol. 2008;54(9):701–17. 35. Burja AM, Banaigs B, Abou-Mansour E, Burgess JG, Wright PC. Marine
cyanobacteria– a prolific source of natural products. Tetrahedron. 2001;57 (46):9347–77.
36. Pauli GF, Case RJ, Inui T, Wang Y, Cho S, Fischer NH, et al. New perspectives on natural products in TB drug research. Life Sci. 2005;78(5):485–94. 37. Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J
Med Res. 2004;120(4):290–304.
38. Falkinham 3rd JO. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev. 1996;9(2):177–215.
39. MacKintosh C, Beattie KA, Klumpp S, Cohen P, Codd GA. Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett. 1990;264(2):187–92. 40. Zegura B, Sedmak B, Filipic M. Microcystin-LR induces oxidative DNA damage in
41. Mandal SM, Barbosa AEAD, Franco OL. Lipopeptides in microbial infection control: scope and reality for industry. Biotechnol Adv. 2013;31(2):338–45. 42. Gerard J, Lloyd R, Barsby T, Haden P, Kelly MT, Andersen RJ. Massetolides A-H,
antimycobacterial cyclic depsipeptides produced by two pseudomonads isolated from marine habitats. J Nat Prod. 1997;60(3):223–9.
43. De Souza Votto AP, Renon VP, Yunes JS, Rumjanek VM, Marques Capella MA, Neto VM, et al. Sensitivity to microcystins: a comparative study in human cell lines with and without multidrug resistance phenotype. Cell Biol Int. 2007;31(11):1359–66.
Submit your next manuscript to BioMed Central and take full advantage of:
• Convenient online submission • Thorough peer review
• No space constraints or color figure charges • Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution
Submit your manuscript at www.biomedcentral.com/submit
